Relationship between protein stability and functional activity in the presence of macromolecular crowding agents alone and in mixture: An insight into stability-activity trade-off Sumra Shahid, Faizan Ahmad, Md. Imtaiyaz Hassan, Asimul Islam * Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India article info Article history: Received 15 July 2015 Received in revised form 19 August 2015 Accepted 23 August 2015 Available online 29 August 2015 Keywords: Macromolecular crowding Thermal stability Enzyme activity Excluded volume effect Stability-activity trade-off Crowding agent abstract The cellular environment is crowded with different kinds of molecules with varying sizes, shapes and compositions. Most of the experiments studying the nature and behaviour of a protein have been done on the isolated protein in dilute buffer solutions which actually do not imitate the in vivo situation. To understand the consequences of such crowded environment, we investigated the effect of macromo- lecular crowding on the stability and activity of hen egg white lysozyme. Two crowding agents, dextran 70 and ficoll 70 which have different shapes and composition, have been employed in this study. To mimic the cellular condition from physiological point of view, the effect of mixtures of both the crowding agents has been also studied. The results indicate that owing to volume exclusion, lysozyme is stabilized while its activity decays with the increasing concentration of both the crowders elucidating the hy- pothesis of stability-activity trade-off. Mixed macromolecular crowding exerts greater effect than the sum of constituent crowding agents (dextran 70 and ficoll 70). © 2015 Elsevier Inc. All rights reserved. 1. Introduction For years, the nature of isolated proteins has been measured in dilute buffer solutions. It has been assumed that this in vitro con- dition mimics the in vivo picture. On the contrary, the cellular environment contains large biomolecules (proteins, nucleic acids, ribosomes and carbohydrates) whose total concentration ranges from 80 to 400 mg ml 1 [1e4]. Such a cellular condition has been termed as macromolecular crowding [5]. This term, when applied to biological systems, describes the fact that inside cells, the total concentration of macromolecules is so high that a meaningful proportion of the volume is physically occupied and, hence, un- available to other molecules [6]. Thus, it is expected that in the non- ideal crowded solutions the behaviour of a test molecule will be different from the diluted solutions [7]. This implies that in the cell protein's physico-chemical properties may be influenced by steric repulsions of macromolecules (volume exclusion). That is, this crowding could affect proteins structure and thermodynamic sta- bility, its function and dynamics and kinetics of protein folding. Thus, it is essential to carry out the studies of biochemical processes in the environment that tries to mimic the intracellular milieu. Many experiments show that protein stability is increased in the presence of macromolecular crowding agents [8e22], and various important studies on the effect of crowding on the enzyme activity have been done [23e35]. In accordance with the assumptions based on excluded volume effects [36], although to different de- grees, all proteins are thermally and chemically stabilized by the presence of crowding agents [37e47]. The excluded volume effect due to the presence of crowding agents was indicated to be the chief player in regulating enzymatic behaviour and stability of proteins. Some of the previous studies have demonstrated that the excluded volume brings about a decrease of both Michae- liseMenten constant, K m , and catalytic constant, k cat , when the enzymatic reaction follows the MichaeliseMenten mechanism [5,23]. Although, there was a distinctive effect of the crowding agent on k cat : where in some cases, k cat was increasing [30,48e52], and in other cases it was decreasing [5,52e54], a slight decrease in K m has been frequently found, regardless of the properties of the crowding agent [25,48e50,53,54]. Abbreviations: GdmCl, guanidinium chloride; UV, ultra-violet; DC p , constant- pressure heat capacity change; T m , midpoint of denaturation; DH m , enthalpy change at T m ; DG D  , Gibbs free energy change at 25  C; K m , Michaelis constant; k cat , cata- lytic constant. * Corresponding author. E-mail address: aislam@jmi.ac.in (A. Islam). Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi http://dx.doi.org/10.1016/j.abb.2015.08.015 0003-9861/© 2015 Elsevier Inc. All rights reserved. Archives of Biochemistry and Biophysics 584 (2015) 42e50